Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.     

Effect of aminoacylation on tRNA conformation

Translational diffusion coefficients have been simulated for various conformations of tRNA^Phe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes.     

Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase

It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.     

Effect of cadmium ions on the rate of the tRNA aminoacylation reaction

Cadmium ions are studied for their effect on the reaction rate of tRNA aminoacylation which was carried out using overall preparations of aminoacyl-tRNA-synthetases (ARSases) isolated from muscles of male rabbits and from three-day pea seedling roots. Cadmium in the concentration of 3 X 10(-5) M is established to accelerate the reaction rate two times as compared to the norm. Other bivalent cations of metals lack this ability.     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

Inhibition of the aminoacylation of selected tRNA molecules by an estrogen-regulated factor on uterine ribosomes. Regulation of aminoacylation of tRNA by estrogens

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen-deprived rats. The extracted inhibitor or a native inhibitor-ribosome complex affects the rate of the peptide elongation reaction in a uterine cell-free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl-tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro greater than Val greater than Arg greater than Try greater than Leu greater than Glu greater than Ile greater than Gly greater than His greater than Ser greater than Lys. Inhibition results from a specific dose-dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl-tRNA synthetase. The effect does not result from removal of A-C-C terminal nucleotides from the 3' end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl-tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol-induced inactivation of a ribosome-associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl-tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl-tRNAs to become rate-limiting in the sequential elongation of peptides.     

Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.

We describe the use of a gel electrophoretic method for measuring the levels of aminoacylation in vivo of mutant Escherichia coli initiator tRNAs, which are substrates for E. coli glutaminyl-tRNA synthetase (GlnRS) due to an anticodon sequence change. Using this method, we have compared the effects of introducing further mutations in the acceptor stem, at base pairs 1:72, 2:71, and 3:70 and discriminator base 73, on the recognition of these tRNAs by E. coli GlnRS in vitro and in vivo. The effects of the acceptor stem mutations on the kinetic parameters for aminoacylation of the mutant tRNAs in vitro are consistent with interactions seen between this region of tRNA and GlnRS in the crystal structure of tRNA(Gln). GlnRS complex. Except for one mutant, the observed levels of aminoacylation of the mutant tRNAs in vivo agree with those expected on the basis of the kinetic parameters obtained in vitro. We have also measured the relative amounts of aminoacyl-tRNAs for the various mutants and their activities in suppression of an amber codon in vivo. We find that there is, in general, a good correlation between the relative amounts of aminoacyl-tRNAs and their activities in suppression.     

Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii.

The genes for 22 tRNA species from Acholeplasma laidawii, belonging to the class Mollicutes (Mycoplasmas), have been cloned and sequenced. Sixteen genes are organized in 3 clusters consisting of eleven, three and two tRNA genes, respectively, and the other 6 genes exist as a single gene. The arrangement of tRNA genes in the 11-gene, the 3-gene and the 2-gene clusters reveals extensive similarity to several parts of the 21-tRNA or 16-tRNA gene cluster in Bacillus subtilis. The 11-gene cluster is also similar to the tRNA gene clusters found in other mycoplasma species, the 9-tRNA gene cluster in M.capricolum and in M.mycoides, and the 10-tRNA gene cluster in Spiroplasma meliferm. The results suggest that the tRNA genes in mycoplasmas have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to mycoplasmas and B.subtilis. The anticodon sequences including base modifications of 15 tRNA species from A.laidlawii were determined. The anticodon composition and codon-recognition patterns of A.laidlawii resemble those of Bacillus subtilis rather than those of other mycoplasma species.     

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.     

Effects of a conformational change in tRNA on its aminoacylation capacity: a study using enzymatically reconstructed variants of T. utilis tRNATyr

Variants of T. utilis tRNATyr containing deletion or substitution of the "conserved" sequence Gm18-G19 in the D-loop have been enzymatically reconstructed in vitro. Conformational analyses of these variants by measuring melting profiles, electrophoretic mobility in "native" polyacrylamide gels, and by the analysis of RNase T1 digestion patterns on sequencing gels laid a stress on the significance of the Gm18-G19 sequence for the maintenance of L-shaped tertiary structure of tRNATyr. Aminoacylation assays with the variant tRNAs at various temperatures revealed that the highly ordered tertiary structure is needed for full aminoacylation capacity.     

Recognition between tRNAs and aminoacyl-tRNA-synthetases of different specificities]

A few examples of incorrect interactions between aminoacyl-tRNA-synthetases and tRNAs extracted from the same organism have already been demonstrated. These interactions can lead, in most cases, to incorrect aminoacylations. The lack of specificity of the aminoacyl-tRNA suggests that incorrect interactions could be a general phenomenon. The aim of this study is to check whether incorrect interactions are a general feature, i.e. whether every aminoacyl-tTNA-synthetase is able to interact with homologous non-cognate tRNAs. In that case, it is interesting to know whether a given aminoacyl-tRNA-synthetase is able to recognize any tRNA or only a particular group of tRNAs. The existence of such groups would lead to the concept of tRNA families. For that, we estimated the affinities of non-cognate homologous tRNA species for yeast valyl-tRNA-synthetase by using competition experiments. The measured affinities varied, in standard aminoacylation conditions, between 1:100 to 1:1000 of that of the non-cognate tRNA. In the absence of Mg2+ ions or in the presence of low concentration of this cation, the affinities were higher and could reach 1:3 of the affinity of the cognate tRNA. On the other hand, we determined the inhibitory effect of a high concentration of tRNAVal toward the aminoacylation of tRNAs specific for 13 amino acids. In order to compare the effects, we determined approximate Km/Ki values. These values ranged from 0.07 for methionyl tRNA synthetase to 0.002 for leucyl tRNA synthetase. For some aminoacyl-tRNA-synthetases, the inhibition was too low to be detected by this technique. Two conclusions arise from this study. First, it seems that non-specific recognitions are quite a general phenomenon. Secondly, if one classifies tTNAs according to their affinities for valyl-tRNA-synthetase, it does not appear any well cut group of tRNAs. This result is not conflicting with the fact that on the basis of aminoacylation criteria several authors have found tRNA and aminoacyl-tRNA-synthetase families since we have already shown that discrimination depends rather on the maximal velocity of the reaction than on the affinity between the tRNA and the aminoacyl-tRNA-synthetases. Finally, the non-existence of clear-cut recognition families of tRNAs casts some doubts on the approach consisting in the characterisation of recognition sites of the tRNAs by the aminoacyl-tRNA-synthetases by comparing the sequences of tRNAs which are amonoacylated by a given aminoacyl-tRNA-synthetase.